Three different types of separation techniques are currently utilized in removing various substances from biological fluids such as blood. These separation techniques are based on different separation principles: (1) mechanical methods (density, size); (2) physical/chemical methods (solubility, electrical mobility); and (3) biological methods (substrate affinity, substrate reactivity).
With regard to mechanical techniques, centrifugal methods are commonly employed in various blood separator systems. Centrifugal methods are currently used in blood banking operations, and more recently for therapeutic plasma exchange. Filtration methods have also been employed for plasma separation and for differential filtration of plasma.
With regard to physical/chemical techniques, precipitation methods can be used employing reduced temperatures, i.e. in the separation of cryoglobulins. High ionic strength methods may also be used for the precipitation of globulins as in the case of the use of ammonium sulfate to precipitate immunoglobulins. Other additives, such as polyethylene glycol, can also be used for precipitation, in particular, the precipitation of immune complexes. An example of an electrical mobility method is the technique of forced flow electrophoresis for the separation of gamma globulin fraction from plasma. Combinations of these methods are used in the Cohn plasma fractionation process which is the major process by which plasma products are obtained.
Biological methods which employ the now-universal concept of biological specificity are the most recent techniques to be developed. Substrate binding involves the "lock and key" concept, and is exemplified by the specificity of the antigen-antibody reaction. Certain antigens and antibodies have been immobilized onto solid support systems and employed in treatment regimes and diagnosis in experimental animals and man. Protein A, derived from certain species of Staphylococcus aureus, binds to IgG and has also been used in macromolecular separation systems. Enzymes exhibit specific substrate reactivity and also have been immobilized, packaged into columns and used in experimental systems.
In many such systems, for instance when antibodies are coupled to solid surfaces, statistically one third of the binding sites are lost since coupling of one of the reactive arms results in concomitant loss of activity of that arm. Stereochemical considerations result in additional losses of binding sites resulting in overall losses of activity of as much as 50%. In addition to these problems, it is necessary to have a separate system for every immunospecific substance to be removed. That is, these systems are not universal in the sense that any immunospecifically recognizable component can be removed by a given system since only those substances which are specific for the bound antigen, antibody, protein, etc. are ultimately removed.
The various separation techniques described above can be further subdivided into off-line and on-line systems. Off-line systems include systems for commercial fractionation of donor plasma, exemplified by the aforementioned Cohn fractionation process, and new techniques such as polyethylene glycol precipitation of immune complexes and low temperature precipitation of cryoproteins. Off-line techniques are particularly useful when separation methods are slow, when there is a need to use toxic additives or when multiple steps in the separation process create difficult control situations.
On-line plasma treatment systems can be divided into three areas based on the degree of recycling involved. Plasma exchange techniques are those in which there is no plasma recycling. These separation techniques result in the separation of blood cells from plasma. Centrifuges in the form of cell separators are commonly used in this technique, along with the more recently developed membrane separation systems. One advantage of this separation technique is that the exact nature of the substance to be removed need not be known since all the plasma is removed. However, this technique requires total replacement of the plasma, and such replacement fluids are prohibitively expensive.
A partial plasma recycling system would involve the removal of a fraction, i.e. globulins, by precipitation, and would only require partial replacement of fluid and/or protein component. An advantage of the system would be a significant reduction in quantity and cost of replacement fluids.
Total plasma recycling involves specific removal of a target substance from the plasma while returning all remaining plasma components to the donor. Although such a system is highly desirable, it is generally applied in the treatment of disease and requires a precise knowledge of the pathogonesis of the disease and the rationale of the treatment.
A more sophisticated and elegant system would be a universal system in which biological fluids, such as whole blood or bone marrow, could be treated for selective removal of the target substance. Treatment of whole blood for selective removal of such substances would, however, require control of coagulation, platelet activation and related activation systems which may interfere with the filtration and/or selective removal of constituents from such biological fluids.